Gene therapy is considered as a new healer of modem medicine since genome sequencing is nearly completed (Anderson, 1992). Numerous methods for gene therapy have been developed in recent years (Mulligan, 1993). Gene therapy vectors used in current clinical trials can be divided into two groups: viral vectors such as retroviruses, adenoviruses or adeno-associated viruses (AAV) and nonviral vectors such as liposomes or naked DNAs (Friedmann, 1999). The most critical parameter of gene therapy is the efficiency of delivery of therapeutic genes to the recipient cells. To meet this goal, vectors need to not only specifically target recipient cells but also stably express therapeutic genes so that the therapeutic effect can be achieved. Lack of tissue-specificity and lack of long-term stable expression are serious drawbacks of current gene therapy vectors (Crystal, 1995).
To obtain efficient delivery of transgenes to target cells, viral vectors are frequently employed for gene therapy protocols. In particular, vectors that are used most often are those derived from retroviruses, adenoviruses or adeno-associated viruses (Crystal, 1995). These viral vectors are nonpathogenic and are designed to be replication-incompetent in recipient cells.
Most attempts to use viral vectors for gene therapy have relied on either retrovirus vectors or adenovirus vectors. Retroviral vectors are capable of maintaining stable gene expression because of their ability to integrate into the cellular genome. However, the disadvantages of retroviral vectors are becoming increasingly clear, including their tropism for dividing cells only, the possibility of insertional mutagenesis upon integration into the cell genome, decreased expression of the transgene over time and the possibility of generation of replication-competent retroviruses. On the other hand adenoviruses can infect nondividing cells, but can induce only transient expression of therapeutic genes. Further, repetitive administration of adenoviral vector to obtain long-term expression frequently induces severe inflammation (Yang et al., 1995). Evidently, these viral vectors need significant improvement before clinical use.
Although these viral vectors are most frequently used, they have a few unacceptable drawbacks. To improve the lack of tissue specificity, targeted viral vectors have been studied in laboratories (Douglas et al., 1999). However, it is not clear whether targeted viral vectors can be clinically used in the near future.
Regarding liver-directed gene therapy, the protocol for these viral vectors are by and large limited to ex vivo therapy, since these vectors lack tissue-specificity (i. e., hepatocyte-specificity). Ex vivo liver-directed therapy involves the surgical removal of liver cells, transduction of the liver cells in vitro (e. g., infection of the explanted cells with recombinant viral vectors) followed by injection of the genetically modified liver cells into the liver or spleen of the patient. A serious drawback for ex vivo liver-directed gene therapy is the fact that hepatocytes (i. e., liver cells) cannot be maintained and expanded in culture. Besides the technical difficulties and complexities, costs involved in each protocol are evidently astronomical.
Ideally, liver-directed gene therapy would be achieved by in vivo transfer of vectors which specifically target hepatocytes. Vectors derived from hepatotropic viruses, such as hepatitis B viruses (HBV), can be administered via circulation and target hepatocytes using the same receptor as the wild-type virus. However, the hepatitis B viruses have not been explored as a gene therapy vector due to lack of information on cis-acting elements essential for HBV genome replication.
Hepatitis B virus (HBV) is the prototype of the hepadnaviridae, a family of a small enveloped DNA virus with pronounced host and tissue specificity (Ganem, 1996). Hepadnaviruses have been found in mammals, e.g., human (HBV), woodchuck (WHV) and ground squirrels (GSHV), as well as in birds, e. g., Pekins ducks (DHBV) and grey herons (HHBV).
One of the bottlenecks in developing an HBV-derived gene therapy vector was a lack of information on cis-acting elements that are essential for viral genome replication. Thus, it is prerequisite to map cis-acting elements across the entire HBV genome.